It is key to any analysis of a biological sample that the integrity of its constituents is conserved between the time that the sample is extracted from a living organism and the time that the analysis is carried out. Sample degradation, however, is both hard to impede, and hard to detect. The result is that many analyses are unable to detect the presence of species that have degraded long before the analysis is carried out; correspondingly, such analyses may in fact identify degradation products of critical components in place of the original components.
The assemblage of proteins in an organism is the key to understanding physiology, function and disease. Proteins are found in many different cellular compartments, for example, in cell nuclei, organelles, protoplasm, and membranes, as well as the inter-cellular space, and in body fluids such as blood. Despite their ubiquity, proteins are extremely sensitive to their environments and thus are not always easy to detect and to identify because they can degrade very quickly.
The natural functions of the assemblage of proteins in an organism are maintained by a complex but delicate balance of biochemical pathways while the organism is alive. Once an organism dies, or once a sample of tissue is extracted from a living organism, the regulatory balance of the organism or in the sample is lost and key proteins start to break down. The breakdown can manifest itself in a number of different ways. For example, some proteins whose natural role is to digest other proteins (a “proteolytic” function), and whose natural levels and activities are regulated while an organism is alive, may go out of control after death. Thus, many proteins and key polypeptides such as coactivators, hormones, and corepressors, end up being actually digested by naturally occurring proteolytic proteins in the sample. Digestion typically involves a rupturing of the polypeptide backbone at one or more points, thereby resulting in protein or peptide fragments. Still other proteins may naturally decompose by other means, such as hydrolysis; whereas in a living organism their levels are maintained because they are continually synthesized, after death they rapidly disappear. For example, post-mortem activity of proteases and oxidative stress has been shown to play an important role on peptide and protein concentration in the brain, as well as for detecting post-translational modifications (Sköld et al., “A Neuroproteomoic Approach to Targeting Neuropeptides in the Brain”, Proteomics, 2, 447-454, 2002; Svensson et al., “Peptidomics-Based Discovery of Novel Neuropeptides”, Proteome Res., 2, 213-219, 2003), both of which are incorporated herein by reference.
For purposes of protein identification, however, to determine what proteins are present in a sample, it is sufficient to be able to ascertain their respective primary structures, i.e., sequences. Proteins and polypeptides have been widely investigated by methods such as two dimensional gels and mass spectrometry, but such techniques depend on having access to samples in which natural protein degradation has not advanced to a point where the concentrations of critical species have been reduced below the various measurement thresholds.
Many proteins undergo natural post-translational modification as part of regulation and modification of their function and activity. Post-translational phosphorylation and de-phosphorylation of proteins is a biological process important for the regulation of cellular processes and signalling. The identification and determination of the level of protein phosphorylations is therefore of great importance for the understanding of protein function and cellular processes.
To study proteins and peptides, tissue or cell samples are usually disrupted by homogenization in certain specific buffer conditions. These buffers often contain ingredients that are supposed to cause a cessation of all protein activity, including proteins (proteases) that degrade other proteins. However, the study of tissue samples from patients or model organisms usually exposes the samples to a certain period of oxygen and nutrient depletion before homogenization and protease inactivation occurs.
Consequently, techniques have been developed in the art for attempting to preserve biological samples after extraction and prior to analysis. Examples of such techniques include tissue fixation, which typically involves immersing a sample in an aldehyde solution, and irradiating samples with microwaves (see, e.g., Theodorsson et al., “Microwave Irradiation Increases Recovery of Neuropeptides from Brain Tissues”, Peptides, 11:1191-1197, 1990). Use of aldehyde solutions is problematic because it penetrates tissue slowly, ˜0.5-2 allowing for degradation of macromolecules prior to complete fixation and consequently does not arrest natural degradation of proteins (Fox et al., “Formaldehyde fixation” J. Histochem. Cytochem. 33:845-853, 1985). Microwave irradiation is problematic because it is generally non-uniform, that is, some parts of the sample reach a temperature that is high enough to cause sample breakdown. Furthermore, parts of the sample can reach temperatures above 100° C. creating small holes due to steam eruptions. (See, for example, Fricker et al., “Quantitative Neuropeptidomics of Microwave-irradiated Mouse Brain and Pituitary”, Molecular & Cellular Proteomics, 4:1391-1405, 2005). Furthermore, microwave irradiation has formerly been applied to living (non-human) subjects as part of a sacrificial protocol and thus has yet to be established as a tool for analyzing samples that have been extracted from subjects, both human and non-human.
WO 2007/024185 describes a method for preparing biological samples for analyses that comprises rapid and uniform heating of the sample after extraction to stop enzymatic degradation of the sample. WO 2007/024185 does not address the need for chemical fixation of samples for histological analyses. For histological analyses it is of importance to obtain fixation of the biological sample prior to submitting the sample to sectioning in order to obtain sections maintaining structures representative of the native structures of the tissue from which the sample and section are derived.
Shiurba et al. (“Immunocytochemistry of formalin-fixed human brain tissues: microwave irradiation of free-floating sections”, Brain Research Protocols 2: 109-119, 1998) describe a method for preparing a biological sample for histological analysis combining formalin fixation with subsequent microwave heating. This method does not address the problems related to rapid post-sampling degradation of the sample.
Investigation of protein phosphorylation, e.g. by the use of phosphorylation state-specific antibodies, offers an important tool in investigative and diagnostic pathology. However, full benefit of the application of this technology e.g. in immunohistochemical studies is limited by the rapid loss of phosphorylation in samples before complete stabilization of the sample can be achieved (Mandell, “Phosphorylation state-specific antibodies. Applications in investigative and diagnostic pathology”, Am. J. Pathol. 163: 1687-1698, 2003).
Accordingly, there is a need for reliable methods for preserving the contents and structures of tissue samples prior to analysis in a way that impedes natural degradation of the sample and that provides reliable and reproducible results.
The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge as at the priority date of any of the claims.
Throughout the description and claims of the specification the word comprise” and variations thereof, such as “comprising” and “comprises”, is not intended to exclude other additives, components, integers or steps.